We will identify and localize proteins specifically associated with the microtubules of the mammalian mitotic apparatus. This work is based on our findings to date that a 150K dalton protein is specificly associated with the microtubules of the mammalian mitotic spindle and that two additional proteins of 105K and 235K daltons are components of mitotic spindles directly isolated following taxol stabilization (Zieve and Solomon, (1982) Cell 28: 233-242). The long term objective of this work is to determine how these proteins contribute to the structure and function of the mitotic apparatus. This work will use a combination of biochemical and immunological approaches. Initial experiments are designed to use cell fractionation combined with two-dimensional gel electrophoresis to localize these proteins in the interphase cell and to determine their time of synthesis during the cell cycle. The remainder of the proposal involves immunocytochemical approaches to studying these polypeptides. Procedures have been developed to isolate sufficient amounts of the individual proteins to produce anti-sera in mice. Both the animal sera, containing polyclonal antibodies, and monoclonal antibodies will be used for these studies. Double indirect immunofluorescence microscopy using the specific mouse antibodies directed against the spindle proteins and a rabbit anti-tubulin antibody will be used to localize these proteins in the cell at all stages of the cell cycle. This work will be an important first step in evaluating the motile significance of these proteins. These studies will then be extended using immunoelectron microscopy to determine the ultrastructural localization of these proteins using the colloidal gold staining technique. Finally, as a direct test of the functions of these proteins, the specific antibodies will be introduced into motile mitotic spindles in vitro and in vivo to determine if they interfere with the motility of these structures. These experiments will be the first in an experimental program to investigate the molecular mechanisms of mammalian spindle motility.